1. ATP7A gene therapy in murine models of Menkes disease. Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment is ineffective in a majority of affected individuals and mortality is high. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia (Fig. 1) and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice;86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-beta-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. These findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease. 2. AAV9 gene therapy. Systemic AAV gene therapy could enable safer, less invasive delivery than brain-directed approaches. To refine prospects for clinical translation of gene therapy for Menkes disease, we evaluated transduction efficiency and therapeutic effectiveness of AAV serotype 9, which has a unique capacity to cross the blood-brain and blood-CSF barriers. In a cohort of mo-br mice, we administered 5x1011 viral genome particles of AAV9 harboring the reduced size (rs) human ATP7A cDNA by intraperitoneal injection on day 2 of life. Western blot analysis indicated high rsATP7A transgene expression in skeletal muscle (Figure 2), and transduction of liver, kidney, heart, lung, intestine, cerebral cortex and choroid plexus epithelia was confirmed by quantitative PCR. Median life span doubled in comparison to untreated affected mo-br mice, demonstrating the efficacy of systemic AAV9. The AAV9 serotype may offer even more effective long-term outcomes than AAV5, based on broader neuronal tropism. We also have successfully performed in utero AAV9 gene transfer in E15 mouse embryos, with a view toward treatment of the dappled mouse allele, a large intragenic deletion in Atp7a that results in prenatal lethality. 3. Capsid engineering for choroid plexus-specific AAV transduction. The choroid plexuses are highly vascularized structures that project into the ventricles of the brain. Besides creating the blood-CSF barrier, the polarized epithelia of the choroid plexus produces CSF by transporting water and ions into the ventricles from the blood and secreting a large number of proteins. A number of neurometabolic diseases, such as lysosomal storage disorders, could benefit from a choroid plexus-targeted gene therapy approach, since CSF flow carries molecules throughout the ventricular system into the subarachnoid space, which covers the entire brain surface. Based on our observation that selective transduction of choroid plexus epithelia enabled rescue of the mo-br mouse, we are developing an AAV vector selectively evolved to target these specialized cells, via phage panning. This powerful method has been used to identify viral capsid peptide motifs that allow superior vector homing to specific cells and tissues. Ultimately, we will evaluate the capacity of the CP-specific selectively evolved AAV to refine and improve outcomes in the mo-br mouse. 4. Choroid Plexus-Directed Gene Therapy for Sanfilippo B Syndrome. In collaboration with Dr. Patricia Dickson at UCLA, we have embarked on a study of choroid plexus-directed gene therapy for mucopolysaccharidosis type IIIB (Sanfilippo B syndrome), a devastating neurological disorder caused by N-acetylglucosaminidase (NAGLU) deficiency. Sanfilippo B is a prototype for a broader category of neurometabolic disorder for which brain-directed gene therapy may prove paradigm-shifting, lysosomal storage diseases. No specific therapy is currently available for Sanfilippo B and disease management consists solely of supportive care. The development of enzyme replacement therapy has been hampered by poor mannose 6-phosphorylation of NAGLU when produced in recombinant form. However, Dr. Dicksons laboratory has developed a fusion protein consisting of insulin-like growth factor 2 (IGF2) and NAGLU that independently binds the mannose 6-phosphate receptor and shows superior intracellular uptake by Sanfilippo B fibroblasts compared to wild-type recombinant NAGLU. This is because the IGF2 moiety binds the mannose 6-phosphate receptor independently of mannose 6-phosphorylation. Intrathecal delivery of recombinant enzyme (injecting enzyme into the cerebrospinal fluid during a spinal tap) has been successful in animal models of other lysosomal storage diseases. However, a major drawback to this approach is the need for repeated (e.g., monthly) intrathecal injections. An alternative route of administration without the need for repeated enzyme injections would be transduction of choroid plexus epithelial cells with an AAV vector containing the cDNA for the enzyme of interest. Since choroid plexus epithelia have an extremely low rate of turnover, this approach will allow a permanent source of enzyme in the CSF for utilization by the brain. Our collaborative studies, recently co-funded by the National MPS Society, will address this issue in detail for Sanfilippo B syndrome.